Allo-HSCT for acute leukemia of ambiguous lineage in adults: the comparison between standard conditioning and intensified conditioning regimens.

Knowledge concerning the clinical and biological characteristics of acute leukemia of ambiguous lineage (ALAL) is limited so that there has been a lack of uniformity in treatment. In this report, we retrospectively investigated the effect of intensified conditioning on adult ALAL undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 59 patients with ALAL (male in 37 cases and female in 22 cases) were consecutively enrolled in the data analyses. Twenty-four patients received the standard conditioning (total body irradiation (TBI) + cyclophosphamide (CY) or busulfan + CY protocol) and 35 received the intensified conditioning (TBI + CY + etoposide or fludarabine + cytarabine plus TBI + CY + etoposide protocol). Five-year transplant-related mortality was 17.6 ± 9.6 % and 25.5 ± 8.0 %, the 5-year overall survival (OS) post-transplantation was 23.8 ± 8.9 % and 64.0 ± 8.4 %, disease-free survival was 16.7 ± 7.6 % and 55.8 ± 9.4 %, the 5-year cumulative incidence of relapse was 80.8 ± 8.5 % and 28.8 ± 9.9 %, respectively, in the standard and the intensified group (P = 0.380, P = 0.029, P = 0.005, and P < 0.001). Both univariate and multivariate analysis indicated that the intensified conditioning regimen and acute graft-versus-host disease were favorable factors to reduce the relapse. The younger patients, patients with CR at the time of transplantation, and the intensified conditioning regimen were favorable factors to elevate the survival. In conclusion, intensified conditioning regimens followed by allo-HSCT might improve long-term survival and decrease relapse of leukemia in adult ALAL compared to the standard conditioning regimens.

[A comparison of toxicity and efficacy between busulfan plus fludarabine and busulfan plus cyclophosphamide for allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia].

OBJECTIVE: To compare the transplant-related toxicity and the efficacy of busulfan/fludarabine (Bu/Flu) and busulfan/cyclophosphamide (Bu/Cy) as conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia(AML) in the first complete remission (CR1). METHODS: Totally 32 AML-CR1 patients underwent allo-HSCT were divided into Bu/Cy (Bu 3.2 mg×kg(-1)×d(-1), 7 - 4 days before transplantation; Cy 60 mg×kg(-1)×d(-1), 3 - 2 days before transplantation) and Bu/Flu (Bu 3.2 mg×kg(-1)×d(-1), 5 - 2 days before transplantation; Flu 30 mg×m(-2)×d(-1), 6 - 2 days before transplantation) groups. The regimen-related toxicity (RRT), incidence and severity of graft-versus-host disease (GVHD), 3-year cumulative relapse rate, non-relapse mortality (NRM), 3-year event-free survival (EFS) rate and overall survival (OS) rate were compared between the two groups. RESULTS: The median follow-up duration was 617.5 (6 - 1261) days. All patients achieved successful engraftment on 30 day after transplantation. There were no significant differences in the median time to neutrophil engraftment (P = 0.121) and platelet engraftment (P = 0.171) between the two groups. The median duration of neutrophil count under 0.1×10(9)/L and platelet count under 20×10(9)/L in the Bu/Cy group were significantly longer than those in the Bu/Flu group (P = 0.000 and P = 0.047). The incidence of grades II-IV RRT were 68.8% and 25.0% (P = 0.032) in the Bu/Cy and the Bu/Flu groups, respectively. There were no significant differences in the incidence of acute GVHD (P = 0.149), chronic GVHD (P = 0.149), incidence of NRM (P = 0.333), 3-year cumulative relapse rates (P = 0.834), 3-year EFS rate (P = 0.362) and OS rate (P = 0.111) between the two groups. CONCLUSION: Compared with Bu/Cy, Bu/Flu is a myeloablative condition regimen with milder bone marrow suppression and lower RRT incidence rate in allogeneic HSCT for AML-CR1 patients without compromising the efficacy.

[Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3].

OBJECTIVE: To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. METHODS: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. RESULTS: The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 µg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 µg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 µg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 µg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 µg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while ß-TG could not. CD36 antibody FA6-152 (25 µg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. CONCLUSION: TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

Long-term outcomes of HLA-matched sibling compared with mismatched related and unrelated donor hematopoietic stem cell transplantation for chronic phase chronic myelogenous leukemia: a single institution experience in China.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative therapy for chronic myelogenous leukemia (CML). In this study, the long-term outcomes of HLA-matched sibling donor (MSD) with mismatched related donor (MRD) and unrelated donor (URD) transplantation for CML in the first chronic phase (CML-CP1) using different graft vs. host disease (GVHD) prophylaxis regimens according to donor source and the degree of HLA matching were compared. The data of 91 patients with CML-CP1 were analyzed with respect to GVHD, overall survival (OS), and transplant-related mortality (TRM). The incidence of grade II-IV acute GVHD was 25.5% in the MSD and 40.5% in the MRD/URD group (P = 0.133). The 1-year cumulative incidence of chronic GVHD was not different between the MSD and the MRD/URD groups, while extensive chronic GVHD was different between the two groups (31.9% vs. 10.8%, P = 0.023). The 5-year cumulative relapse rate was not different between the MSD and the MRD/URD groups, while TRM was different between the two groups (6.6% vs. 26.3%, P = 0.010). The 5-year cumulative OS was 90.9%, 71.5%, and 85.4% in the MSD, the MRD/URD, and the HLA allele-matched URD transplantation, respectively (MSD vs. MRD/URD, P = 0.013; MSD vs. HLA allele-matched URD, P = 0.437). In conclusion, survival in HLA allele-matched URD is equivalent to MSD, but in MRD and mismatched URD is inferior to MSD in patients with CML-CP1 undergoing allo-HSCT using different GVHD prophylaxis regimens according to donor source and degree of HLA matching. Patients undergoing MRD/URD transplantation have an equal quality of life as patients undergoing MSD transplantation.

[Allogeneic hematopoietic stem cell transplantation for acute leukemias of ambiguous lineage in adults: a comparison between the conditioning of different intensities].

OBJECTIVE: To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the conditioning of different intensities for acute leukemias of ambiguous lineage (ALAL). METHODS: A total of 38 ALAL patients were treated with two conditioning of different intensities in our hospital from March 2002 to August 2010. The standard conditioning included TBI + Cy or Bu + Cy, intensified conditioning included Fludarabine + Ara-C + TBI + Cy. Cyclosporine A (CsA) and methotrexate (MTX) were administered in patients with human leukocyte antigen-matched sibling donor. And CsA, MTX plus antihuman thymocyte globulin and/or mycophenolate were used in all patients with HLA-mismatched related donor and unrelated donors transplants for graft-versus-host disease (GVHD) prophylaxis. COX regression was used to evaluate the prognostic factors of ALAL. RESULTS: Among 38 ALAL patients, 19 received the standard conditioning while another 19 the intensified conditioning. All patients achieved hematopoietic reconstitution. The 5-year overall survival (OS) and the disease-free survival (DFS) were 35.5% and 25.7% respectively. The 5-year OS rates were 20.2% and 48.1% (P = 0.233) and DFS 6.5% and 43.1% (P = 0.031) in the standard and intensified conditioning groups respectively. The 5-year cumulative relapsing incidence was 58.9% in all patients and 87.6% vs 30.4% in the standard and intensified conditioning groups respectively (P = 0.003). Through a COX regression model for univariate analysis, the intensified conditioning and chronic GVHD were protective factors for DFS (P = 0.001, 0.031). CONCLUSION: The intensified conditioning in ALAL patients undergoing allo-HSCT may improve the long-term patient survival and decrease the relapse of leukemia. The graft versus leukemic effect has some efficacy in ALAL patients undergoing allo-HSCT.

[Clinical Characteristics and Prognosis of Elderly Patients with Medium and High risk Myelodysplastic Syndrome].

OBJECTIVE: To investigate the clinical characteristics and prognosis of patients with medium and high risk myelodysplastic syndrome (MDS). METHODS: 97 MDS patients above the age of 60 treated in Nanfang Hospital, Southern Medical University from February 2011 to August 2020 were enrolled. The clinical characteristics and prognosis of the MDS patients with medium risk, high risk or very high risk based on IPSS-R category were retrospectively analyzed. According to the difference of treatment regimes, the patients were divided into the transplantation group, chemotherapy group and other treatment group, and the efficacy among the patients in the 3 groups were analyzed. RESULTS: MDS with excess blast (MDS-EB) in the elderly patients with medium and high risk MDS were the most common, 47.4% of the patients with abnormal chromosome karyotypes, and 23.7% with complex karyotypes (≥3). 97.3% of the patients showed at least one gene mutation, and TP53 mutations were detected in nearly 20% of the patients with medium and high risk. Multivariate analysis showed that IPSS-R category and treatment regimes were the factors affecting the prognosis of elderly patients with medium and high risk MDS. The median overall survival (OS) time of the patients in the 3 groups showed significant difference (P=0.012), and the median OS of the patients in the transplantation group was significantly longer than that in the chemotherapy group and other group (P=0.003,P=0.014,respectively), while there was no significant difference in median OS between chemotherapy group and other treatment group (P=0.685). CONCLUSION: Elderly MDS patients with medium and high risk can benefit from allogeneic hematopoietic stem cell transplantation, which will prolong their OS.

[Correlation between U2AF1 Gene Mutation Characteristics and Clinical Manifestations and Prognosis in Patients with Myelodysplastic Syndrome].

OBJECTIVE: To investigate the correlation between U2AF1 gene mutation and clinical manifestations and prognosis in patients with myelodysplastic syndromes (MDS). METHODS: The clinical data of 203 MDS patients who accepted Next Generation Sequencing (NGS) was retrospectively analyzed in Nanfang Hospital, Southern Medical University from December 2012 to October 2019. According to whether the patients had U2AF1 gene mutation, the patients were divided into U2AF1 mutated group and non-mutated group, and the relationship between gene mutation characteristics and clinical manifestations and prognosis was analyzed. Then according to the difference of the mutation site of U2AF1, the patients in U2AF1 mutated group were divided into U2AF1S34 mutated group and U2AF1Q157/R156 mutated group, and the correlation between gene mutation characteristics and prognosis was analyzed. RESULTS: The incidence of U2AF1 mutation in MDS patients was approximately 11.3% (23/203), and the mutation frequency of U2AF1 allele was 32.5%. The male ratio in U2AF1 mutated group was significantly higher than that in U2AF1 non-mutated group (P=0.001). There was no patient who had complex karyotypes or TP53 gene mutation in U2AF1 mutated group. There were no significant differences in ages, blood parameters, bone marrow blasts, WHO 2016 classification, IPSS-R category, chromosomal abnormalities like del(5q), -7/del(7q), del(20q), +8, and gene mutation like ASXL1, DNMT3A, RUNX1, SF3B1, and SRSF2 mutation between U2AF1 mutated group and the non-mutated group. Compared with the non-mutated group, there was no significant difference in the overall survival time (P=0.377), the time of acute myeloid leukemia (AML) transformation (P=0.681), and the response rate to hypome- thylating agents in U2AF1 mutated group (P=0.556). Besides, no differences were observed in sex, diagnosis age, WHO 2016 classification, IPSS-R category, blood parameters, overall survival time, and AML transformation time between U2AF1S34 mutated group and U2AF1Q157/R156 mutated group. CONCLUSION: The U2AF1 gene mutation dose not affect the survival time, AML transformation time, and response rate to hypomethylating agents in MDS patients. Besides, there are no statistical differences in the clinical characteristics and prognosis of MDS patients between U2AF1S34 mutated group and U2AF1Q157/R156 mutated group. Transplantation shows no significant benefit for patients with U2AF1 mutation.

[The clinical characteristic and risk factors for the incidence of invasive fungal infection in patients following allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To approach the clinical characteristic and risk factors affecting the incidence and outcome of post-transplantation invasive fungal infection (IFI) in patients with malignant hematologic disease and undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The incidence and outcome of IFI after transplantation in 193 cases of recipients underwent allo-HSCT in our single center were assessed retrospectively. Some potential influential factors for that of IFI were analyzed by methods of bivariate correlate analysis and binary logistic regression analysis, including the source of donor and stem cell, human leukocyte antigen matched, white blood cell engraftment, the history and states of IFI before transplantation, prophylaxis schema for GVHD, acute and chronic GVHD. RESULTS: The 2-year cumulative incidence of post-transplantation IFI was 34.0% +/- 4.0%. The incidences of breakthrough IFI at primary and secondary prophylaxis were 3.8% and 21.1%, respectively (P = 0.000). 84.2% patients with IFI occurred within half of years after transplantation. In fungus that can be measured, molds and yeasts accounted for 68.1% and 27.7%, respectively. Infective sites in pulmonary or not accounted for were 67.3% and 27.7%, respectively. The total effective rate of IFI was 67.3%, complete response rate was 44.2%. Furthermore, there was no statistically significant difference in the therapeutic effect of antifungal agents between patients with a history of IFI and those without before transplantation. IFI-related mortality was 38.5%. Muti-variate analysis showed acute GVHD was an important factor affecting the incidence and outcome of IFI. CONCLUSION: Pulmonary mold infection was the most common IFI after allo-HSCT. patients with a history of IFI did not seem to be an contraindication for allo-HSCT. Acute GVHD was a significant risk factor for the incidence and outcome of IFI.

[Detection Rate of Central Nervous System Leukemia Can Be Improved by Cell Preservation Solution].

OBJECTIVE: To investigate whether cell preservation solution can prolong the survival time of leukemia cells and increase the survival rate, so as to improve the detection rate of central nervous system leukemia. METHODS: Kasumi cells were added into cerebrospinal fluid (CSF) supernatant with or without cell preservation solution to compare cell viability and biological characteristics at different time point. Wright Giemsa staining was used to compare cell morphology; cell counting, CCK-8 method, and trypan blue staining were used to compare the cell number, and flow cytometry was used to compare the cell viability. The expression of AML-ETO tumor fusion gene was detected by fluorescence quantitative RT-PCR. RESULTS: At different time points (8 h and 24 h), the survival, molecular biological characteristics and RT-PCR result of the cells in CSF with cell preservation solution were significantly better than those in normal cerebrospinal fluid. CONCLUSION: Cell preservation solution can effectively improve the survival time and survival rate of leukemic cells, thereby increase the detection rate of CNS leukemia.

[Sclerodermatous chronic graft-versus-host disease after hematopoietic stem cell transplantation: incidence, clinical characteristics and risk factors].

OBJECTIVE: To investigate the incidence and risk factors of sclerodermatous chronic graft-versus-host disease (ScGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The clinical data of 259 patients undergoing allo-HSCT in Nanfang Hospital between January, 2012 and December, 2014 were analyzed. RESULTS: Chronic GVHD following allo-HSCT occurred in 134 (51.7%) cases, among whom 22 patients showed sclerodermatous features at a median of 12.5 months (range 4-28 months) after the transplantation. The overall incidence of ScGVHD was 8.49% (22/259) in the recipients and 16.4% (22/134) in those with cGVHD. Univariate analysis showed that the conditioning regimen with total body irradiation (P=0.031), GVHD prophylaxis with MMF (P=0.046), presence of chronic GVHD (P=0.008), and donor lymphocyte infusion (P=0.001) were all closely associated with the occurrence of ScGVHD. Multivariate analysis identified chronic GVHD (RR=3.512, 95%CI: 1.235-9.987, P=0.018) and donor lymphocyte infusion (RR=5.217, 95%CI: 1.698-16.029, P=0.004) as the independent risk factors of ScGVHD. CONCLUSION: ScGVHD following allo-HSCT is not a common complication, and cGVHD and donor lymphocyte infusion are the independent risk factors for ScGVHD.

[Establishment and Applications of Double-Fluorescent Protein Allo-Transplantation Mice Model].

OBJECTIVE: To establish allo-transplantation model by using mRFP⁺ to eGFP⁺ transgenic mice and to observe the distribution of donor cells and donor-recipient cellular interaction in the bone marrow after semi-solid decalcification (SSD). METHODS: After myeloablative irradiation, C57BL/6 female eGFP⁺ transgenic mice were infused with (5 × 106) bone marrow cells from FVB male donor mice through tail vein. The control group was infused with PBS. Then the general conditions, engraftment level, hematopoietic recovery, incidence of GVHD and survival of recipients were evaluated after transplantation. In the recovery process, SSD was used to treat the femora before observing the cells distribution, morphology and interaction by confocal microscopy directly or after making frozen section. RESULTS: WBC of recipient eGFP⁺ mice was recovered on (20 ± 3.07) d, (93.94 ± 1.59)% in peripheral cells were RFP⁺ cells (n = 10), GVHD happened in 4 of 10 mice within 1 month. During SSD, the hard components were replaced gradually and RFP⁺ cells could be seen mainly in the bone trabecula and surrounded by eGFP⁺ cells under confocal microscope, their interactions could be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction. CONCLUSION: The double fluorescent allo-transplantation mouse model successfully established, by means of our novel protocol named SSD, the donor and recipient cell location and their interaction can be visually observed, which provides the basis for clinical studies on the distribution and homing of donor cells, and some related explorations after transplantation.

[Clinical analysis of acute heart failure's risk factor for chronic myelogenous leukemia patient during the early stage of allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: The study was to analyze the acute heart failure's risk factors and clinical characteristics for the patient with chronic myelogenous leukemia (CML) during the early stage (within 100 d) of allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: A total of 106 cases of CML received allo-HSCT were retrospectively studied in Nanfang Hospital from May 2003 to May 2013. On the basis of existence or absence of acute heart failure during early stage of allo-HSCT (100 d), the patients were divided into heart failure (15 cases) and control group (91 cases). Using Logistic univariate analysis, Fisher' exact test and Pearson X(2) test, the acute heart failure's risk factors and clinical characteristics of both groups were analyzed. RESULTS: The median occurrence time of acute heart failure was 3 d (1 d before transplantation to 84 d after transplantation). Logistic univariate analysis indicated that the imatinib treatment history and time, and the prophylaxis regimens for GVHD with anti-thymocyte globulin (ATG) were all the poor prognostic factors for acute heart failure. Incidence of hepatic veno-occlusive disease (HVOD), bacterial infection and adverse prognostic events including death in the heart failure group patients were statistically higher than that in control group (P < 0.05). CONCLUSION: Acute heart failure mostly happened in the early stage after allo-HSCT, imatinib treatment and GVHD prophylaxis regimens with ATG are the poor prognostic factors for acute heart failure. The patients of heart failure group seem to have higher incidence of hepatic veno-occlusive disease (HVOD), bacterial infection and deaths.

Expression and cytotoxicity of EGFP-labeled D-amino acid oxidase in HeLa cells.

To observe the expression of R. gracilis D-amino acid oxidase (DAAO) gene labeled by EGFP in HeLa cells and how it mediates cytotoxicity, DAAO cDNA and EGFP gene were cloned into Eukaryotic expression vector pIRES to construct DAAO gene expression vector pIRES-DAAO. HeLa cells were transfected with pIRES-DAAO in vitro. Expression of EGFP gene in HeLa-D cells was observed under a fluorescent microscope. The efficiency of transfection was analyzed by flow cytometry (FCM). The fluorescent cells were screened by FCM and were named HeLa-D. The activity of HeLa-D cells was detected by MTT colorimetric assay after they were treated with D-Ala at various concentrations. The results indicted that expression of EGFP gene in HeLa-D cells was seen under fluorescent microscope and HeLa-D cells were screened by FCM. Apparently,the prodrug D-Ala killed HeLa-D cells. These results demonstrate that EGFP gene can be regarded as a reporter gene to screen the cells transduced with DAAO quickly, and DAAO/D-Ala suicide gene system may prove helpful in gene therapy of cancer.

[Large-scale real-time titration of green-fluorescence-protein-marked recombinant retrovirus: comparison with standard titration method].

OBJECTIVE: To compare large-scale real-time titration method (LaSRT) with standard titration method by flow cytometry (FACS) for determining the titers of green-fluorescence-protein (GFP)-marked recombinant retrovirus. METHODS: (1) Standard titration method: NIH3T3 cells were inoculated at 2x10(5) /well in 6-well plate, and after cell culture for 12 h, 0.5 ml, 50 microl and 5 microl GFP-marked recombinant retrovirus (n=3) were respectively used to infect the cells, with the final concentration of polybrene being 8 microg/ml. Forty-eight hours later, the cells were treated with trypsin and assayed for the positive rate of GFP by means of FACS. When the positive rate was lower than 10%, the titer was calculated according to the equation: virus titer (TU/ml) =2x10(5)xGFP positive rate/volume of virus stock solution used. (2) LaSRT method: The cells were inoculated at 5,000 cells/well in a 96-well plate, and after cell culture for 12 h, 90 microl/well complete culture medium was used with 8 microg/ml polybrene and 10% newborn bovine serum, 10 microl virus was added into the first well, and ten-fold dilution of the previous virus-containing solution was performed before the virus was added into the next well (8 wells in each group, altogether 3 groups). Forty-eight hours later, inverted fluorescence microscope was used to observe the fluorescence-positive cells in each well. The virus titer was calculated according to the equation: virus titer (TU/ml) =mx10(n+1), where n is the serial number of the reference well, and m the number of positive cells. (3) LaSRT was used to study the influence of freezing/thawing on the titers of recombinant retroviruses. RESULTS: The virus titer obtained with standard method by FACS was (1.54+/-0.38)x10(6) TU/ml, and that of LaSRT was (1.33+/-0.57)x10(6) TU/ml (P>0.05). After one cycle of freezing/thawing, the virus titer dropped to (18.1+/-9.9)% (n=7). CONCLUSION: LaSRT is more rapid and convenient as well as easier to determine the virus titer compared with standard method, and no significant difference is found between the two titration methods.

[Exploration and analysis of PCR-inhibitory components in peripheral blood and bone marrow samples].

PCR is an enabling and preferred technology for the detection of DNA or mRNA in genomic age, and has been widely used in molecular medicine, biotechnology, microbiology and diagnostics. PCR has many excellent traits, such as high specificity and sensitivity, short handle time, reliable and effective results, low demand for sample. In recent years, real-time reverse transcription PCR (qRT-PCR) has acquired an impressive improvement in the detection of mRNA, it has a perfect sensitivity and can quantify some rare mRNA copies as low as less ten. The progress in PCR results in a more strict demand for sample, so it is necessary to take the potential PCR-inhibitory components in peripheral blood and bone marrow samples into account again. This review explores the potential PCR-inhibitory components in peripheral blood and bone marrow samples, and discusses the possible mechanism and solutions for the inhibition effect, also emphasizes the potent effect of the frequently-used anticoagulant heparin sodium in PCR. This article will be helpful to assess the PCR detection results (especially qRT-PCR ) in some degree, for example, can use it to decide whether the PCR detection results are reasonable for some low-expression genes, or use it to eliminate the false negative results generated by PCR-inhibitory components in sample.

Bruton's tyrosine kinase: potential target in human multiple myeloma.

Bruton's tyrosine kinase (BTK), a Tec family non-receptor tyrosine kinase that is required for B cell development, is critical for the initiation and maintenance of human B-cell malignancies. However, the expression of BTK and the role that BTK plays in the pathogenesis of multiple myeloma (MM) remain seldom reported. In this study we examined the expression and screened for gene mutations of BTK in MM cells. We showed that BTK was elevated and activated in a dexamethasone-resistant cell line and in two out of nine (22.2%) patients' cells. Interestingly, patients with higher BTK expression had a poorer prognosis. In addition, a single nucleotide polymorphism (SNP) at cDNA position 2062 (T2062C) in the BTK gene was recorded in six out of eight (75%) patients and in U266 cells. This SNP in MM cells was not detected in other malignant hematopoietic cells of different lineages. These results suggest that the function of BTK warrants further investigation, and BTK expression might be used as a prognostic indicator for patients with MM.

The effect of imatinib therapy on the outcome of allogeneic stem cell transplantation in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia.

OBJECTIVE: To evaluate the efficacy of imatinib administration before and/or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). METHOD: Patients with imatinib therapy time exceeding 30 days pre-/post-transplant were screened in our data. Imatinib was used in induced or consolidated chemotherapy pre-transplant, or maintenance therapy after 60 days post-transplant (therapy time was less than 180 days) regardless of the molecular status of the disease. RESULTS: Sixty-nine patients with Ph+ ALL were enrolled in the retrospective analysis. Forty-four patients received imatinib therapy, including 24 pre-transplant, 9 post-transplant, and 11 both pre- and post-transplant. With a median follow-up time of 395 days (range, 55-2762 days) post-transplant, 3-year estimated overall survival was 62.3 ± 16.6, 40.0 ± 21.9, 41.7 ± 22.2, and 25.9 ± 11.4%, respectively (P = 0.221), and disease-free survival (DFS) was 53.6 ± 17.9, 20.0 ± 17.9, 33.3 ± 25.5% and 23.6 ± 11.4%, respectively (P = 0.421), in patients with imatinib therapy pre-transplant, post-transplant, both pre- and post-transplant, neither pre- nor post-transplant. The incidence of relapse at 3 year for patients with imatinib therapy post-transplant (n = 20) was 63.6%, comparing with 24.2% (P = 0.018) in patients without imatinib therapy post-transplant (n = 49). The ratio of CD4+CD25+Foxp3+ cells in blood was significantly higher at 30 and 60 days after imatinib therapy than that at the time of pre-imatinib in 20 patients (P = 0.019 and 0.001, respectively). CONCLUSIONS: Application of imatinib pre-transplant might have benefited for patients with Ph+ ALL. Whether administration of imatinib, regardless of the molecular status of the disease post-transplant increases relapse, is a worthy goal for further study.

[Anti-apoptotic effect of Astragalus Polysaccharide on myeloid cells].

This study was aimed to assess the effect of Astragalus Polysaccharide (ASPS) on in-vitro hematopoiesis. CFU-GM assays were used to determine the effect of ASPS and thrombopoietin (TPO) on granulocytic-monocyte progenitor cells. The CFU assays were also used to investigate the effect of ASPS on the proliferation of HL-60 cells.HL-60 cells were cultured with serum-free RPMI 1640 medium and treated with or without of different concentrations of ASPS. After 72 h incubation, the number of cells were counted.In addition, the caspase-3 and JC-1 expression was determined by flow cytometry with Annexin V/PI double staining. The results showed that ASPS (100, 200 µg/ml) and TPO (100 ng/ml) significantly promoted CFU-GM formation in vitro. Various concentrations of ASPS and TPO also promoted the colony formation of HL-60 cells, the largest effect of ASPS was observed at a concentration of 100 µg/ml. There were no synergistic effects between TPO and ASPS on cellular proliferation. The results also showed that ASPS significantly protected HL-60 cells from apoptosis in condition of serum-free medium culture, suppressed caspase 3 activation, and reduced the cell apoptosis. It is concluded that ASPS can significantly promote the formation of bone marrow CFU-GM and the proliferation of HL-60 cells, the optimal concentration of ASPS is at 100 µg/ml. In the absence of serum inducing apoptosis, ASPS also significantly reduced the apoptosis of HL-60 cells via suppressing the activation of caspase-3.

[Clinical significance of monitoring BK polyomavirus in patients after hematopoietic stem cell transplantation].

This study was aimed to establish a method for rapid detecting BK polyomavirus (BKV) and to investigate the feasibility and value used in leukemia patients undergoing hematopoietic stem cell transplantation. Primers were designed according to BKV gene sequence; the quantitative standards for BKV and a real-time fluorescent quantitative PCR for BKV were established. The BKV level in urine samples from 36 patients after hematopoietic stem cell transplantation were detected by established method. The results showed that the standard of reconstructed plasmid and real time fluorescent quantitative PCR method were successfully established, its good specificity, sensitivity and stability were confirmed by experiments. BKV was found in 55.56% of urine samples, and the BKV load in urine was 2.46 × 10(4) - 7.8 × 10(9) copy/ml. It is concluded that the establishment of real-time fluorescent quantitative PCR for BKV detection provides a method for early diagnosis of the patients with hemorrhagic cystitis after hematopoietic stem cell transplantation.

[The outcome and safety of mesenchymal stem cells from bone marrow of a third party donor in treatment of secondary poor graft function following allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To evaluate the efficacy and safety of bone marrow-derived mesenchymal stem cells (MSC) from a third party donor for secondary poor graft function (PGF) following allogeneic hematopoietic stem cell transplantation(allo-HSCT). METHODS: Five patients with secondary PGF were treated with MSC at a dose of 1 x 10(6)/kg body weight at a median of 47 days (35 to 61) after secondary PGF. MSC were derived from bone marrow (BM) of HLA-disparate third party donors, cultured in vitro and infused without HSC. If absolute neutrophil cell (ANC) and platelet counts (PLT) did not reach the standardization of > 1.5 x 10(9)/L and > 50.0 x 10(9)/L, respectively, within 28-30 days after the first MSC treatment, a second MSC treatment was required. RESULTS: MSC were infused once in one patient and twice in four patients with an interval of 28 to 30 days. All patients obtained ANC and PLT recovery at a median of 34 (25 to 49) days and 47 (26 to 54) days, respectively, without toxic side effects within follow-up periods of median 761 (204-1491) days. Three patients developed Epstein-Barr virus (EBV) reactivation at 42, 48, 108 days after MSC infusion, respectively and two of the three coverted to posttransplant lymphoproliferative disorders (PTLD). CONCLUSION: MSC from a third party donor are effective to patients with secondary PGF following allo-HSCT, whether it might increase the risk of EBV reactivation and EBV-associated PTLD need further observation.